Research Article Received: 22 September 2014
Revised: 1 October 2014
Accepted: 2 October 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 2729–2734 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7068
Radical-directed dissociation of peptides and proteins by infrared multiphoton dissociation and sustained off-resonance irradiation collision-induced dissociation with Fourier transform ion cyclotron resonance mass spectrometry Xing Zhang1, Huilin Li3, Benjamin Moore1, Piriya Wongkongkathep2, Rachel R. Ogorzalek Loo3,4, Joseph A. Loo2,3,4** and Ryan R. Julian1* 1
Department of Chemistry, University of California, Riverside, CA 92521, USA Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA 3 Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA 4 UCLA/DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095, USA 2
RATIONALE: Recent experiments utilizing photodissociation in linear ion traps have enabled significant development of Radical-Directed Dissociation (RDD) for the examination of peptides and proteins. The increased mass accuracy and resolution available in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) should enable further progress in this area. Preliminary experiments with photoactivated radicals are reported herein. METHODS: A 266 nm Nd:YAG laser is coupled to a FTICR or linear ion trap mass spectrometer. Radical peptides and proteins are generated by ultraviolet photodissociation (PD) and further activated by collisions or infrared photons. RESULTS: A 266 nm UV laser and an IR laser can be simultaneously coupled to a 15 Tesla FTICR mass spectrometer. The ultra-low-pressure environment in FTICR-MS makes collisional cooling less competitive, and thus more secondary fragments are generated by UVPD than in linear ion traps. Activation by sustained off-resonance irradiation collisioninduced dissociation (SORI-CID) or infrared multiphoton dissociation (IRMPD) also yields additional secondary fragmentation relative to CID in an ion trap. Accurate identification of RDD fragments is possible in FTICR-MS. CONCLUSIONS: Relative to linear ion trap instruments, PD experiments in FTICR-MS are more difficult to execute due to poor ion cloud overlap and the low pressure environment. However, the results can be more easily interpreted due to the increased resolution and mass accuracy. Copyright © 2014 John Wiley & Sons, Ltd.
Bottom-up proteomics is widely used for protein identification and de novo protein sequencing.[1] Tandem mass spectrometry (MS/MS) can effectively verify the sequences of peptides with high confidence, speed and sensitivity, particularly with the aid of a high-performance mass spectrometer. Collisioninduced dissociation (CID) is the most widely used dissociation method for peptide/protein characterization due to its high efficiency and ease of implementation.[2] Electron-based dissociation methods, including electron-capture dissociation (ECD) and electron-transfer dissociation (ETD), have also proven to be effective in peptide sequencing and especially for the identification of post-translational modifications (PTMs).[3] The fragmentation types in these electron-based methods are complementary to the vibrational excitation dissociation pathways accessed by CID and thus can
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* Correspondence to: R. R. Julian, Department of Chemistry, University of California, Riverside, CA 92521, USA. E-mail: [email protected] ** Correspondence to: J. A. Loo, Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA. E-mail: [email protected]
potentially provide other useful structural information not available from CID. In contrast to even-electron dissociation pathways that dominate in CID, many of the fragments in ECD and ETD are generated by radical chemistries that initiate various homolytic bond cleavages. In addition to electron-based methods, radicals can also be generated on polypeptides in a more controlled manner through modification with radical precursors. Various radical precursors and methods have been developed to generate hydrogendeficient radical peptides in the gas phase, including oxidative dissociation of peptide-metal complexes,[4–6] and homolytic bond cleavage in modified groups such as nitrate ester,[7] peroxycarbamate,[8] nitroso,[9] and TEMPO.[10] These methods use CID to facilitate activation of radical precursors containing labile bonds that generate hydrogen-deficient radicals upon dissociation. In addition, photodissociation (PD) can be used for radical initiation, which offers the advantage of selective activation of specific chromophores.[11–13] Ultraviolet photodissociation (UVPD) at 266 nm has been used to selectively cleave carbon–iodine (C–I) or carbon–sulfur bonds that are present in chromophore-modified peptides and proteins or native sulfur–sulfur bonds present in disulfide bridges. After generation of radical peptides, further dissociation (either
X. Zhang et al. spontaneously or aided by collisional activation) leads to radicaldirected dissociation (RDD), including backbone fragmentation and side-chain loss. One typical feature in RDD is relatively abundant side-chain loss, which allows unambiguous identification of many specific amino acid residues and even distinction of isobaric residues such as leucine and isoleucine.[14] Both ion trap (linear and quadrupole) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers have the capability to trap ions for a flexible amount of time, which makes them ideal for coupling with lasers for photodissociation experiments. A variety of lasers with different wavelengths have been implemented on quadrupole ion trap[15–22] and FTICR mass spectrometers.[23–26] Although both instruments are capable of trapping ions, there are also important and obvious differences between them. For example, FTICR instruments are capable of achieving much higher resolution and mass accuracy than ion traps. There are also significant differences in pressure, with FTICR cells operating at 6–7 orders of magnitude lower pressure than ion traps. The lower pressure of the FTICR cell is advantageous for infrared activation of ions because there is minimal cooling.[27] On the other hand, FTICR cells are much larger than ion traps, require more time per scan, and they are located in the center of a large cryogenic magnet, which makes optimization of laser alignment including maximizing laser/ion cloud overlap significantly more challenging than interfacing lasers to linear ion traps. In the present work, 266 nm Nd:YAG lasers are coupled to both FTICR and linear ion trap (LIT) mass spectrometers. UVPD is performed on trapped ions in both instruments to cleave C–I bonds. The ions are then subjected to further activation by CID or infrared multiphoton dissociation (IRMPD) to investigate their subsequent radical chemistry. The differences in radical-directed dissociation between FTICR and LIT instruments are examined.
EXPERIMENTAL Materials A synthetic peptide with amino acid sequence RGYALG (residues 21-26 of chicken lysozyme and a synthetic peptide substrate for a cyclic-AMP dependent protein kinase[28]) was purchased from the American Peptide Company (Sunnyvale, CA, USA). Sodium iodide, chloramine-T, sodium metabisulfite and other organic solvents were purchased from Thermo Fisher Scientific (Waltham, MA USA). Bovine ubiquitin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Peptide derivatization Peptides were labeled at the N-terminus with N-hydroxysuccinimide (NHS)-activated iodobenzoyl ester (2× molar ratio) in 1:3 borate buffer (0.2 M, pH 8.5)/dioxane solution for 30 min at 40 °C. The side products at the arginine and tyrosine side chains were removed by incubation in 1 M hydroxylamine (adjust pH to 8–9 with NaOH) for 4 h. Protein iodination
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The tyrosine residue in ubiquitin (Y59) was iodinated with sodium iodide and chloramine-T at room temperature. After 10 min reaction time, excess sodium metabisulfite was added
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to quench the reaction. Stoichiometric quantities of reagents (1:2:1:2 molar ratio of peptide/sodium iodide/chloramine-T/ sodium metabisulfite) were used to limit the iodination extent and produce mainly mono-iodinated tyrosine. A reversed-phase cartridge (Michrom Bioresources, Inc., Auburn, CA, USA) was used for peptide and protein desalting and purification. The samples were washed with 2% acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA) and eluted with 80% ACN with 0.1% TFA. Photodissociation MS in linear ion trap instruments The polypeptides (10 μM) were dissolved in 50:50 H2O/CH3OH with 0.1% acetic acid and electrosprayed into an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). 266 nm photons were generated from the 4th harmonic of a Nd:YAG laser (Continuum, Santa Clara, CA, USA). The back plate of the instrument was modified with a quartz window to transmit UV photons into the linear ion trap. Laser pulses were synchronized to the isolation step via a digital delay generator. Photodissociation MS in FTICR-MS The experiments were performed using a 15-Tesla Bruker SolariX FTICR mass spectrometer with an infinity cell (Bruker Daltonics, Bremen, Germany). Data were acquired with an approximate resolving power of 400 000 at m/z 400. The electrospray ionization (ESI) capillary voltage was set to 4 kV. The temperature of dry nitrogen was set to 180 °C and the flow rate was kept at 2.5 L/min. The radiofrequency (RF) amplitude of the ion funnels was 200 Vpp, and the applied voltages were 100 V and 6 V for funnels 1 and 2, respectively. The voltage of skimmer 1 was 20 V and the skimmer 2 voltage was kept at 5 V. The RF frequencies used in all ion-transmission regions were multipole 1 (5 MHz), quadrupole (2 MHz), and transfer hexapole (2 MHz). Ions were accumulated for up to 20 s in the hexapole collision cell before being transmitted to the infinity ICR cell. The time-of-flight was set to 1.5 ms. The vacuum pressures for different regions were ~2 mbar for the source region, ~2 × 10–6 mbar for the quadrupole region, and ~2 × 10–9 mbar for the UHV-chamber pressure. IRMPD was performed with a Synrad 30 W CO2 laser (Mukilteo, WA, USA). Firing of the laser pulses (266 nm, Continuum Nd:YAG laser) was controlled by custom-programmed digital signals from the FTICR mass spectrometer. To align both UVPD and IRMPD lasers at the back of the infinity cell, a fused-silica window (Esco Optics, Oak Ridge, NJ, USA), positioned as a beam splitter, reflected the 10.6 μm beam 90° to the cell while transmitting the UV beam to the infinity cell. The experiment proceeded as follows: ions were filtered by m/z value in the quadrupole (Q1) and accumulated in the hexapole collision cell (h), then submitted to the infinity cell. One to ten UV pulses (10 Hz) homolytically cleaved carbon–iodine bonds. The radical species produced were isolated in the infinity cell (pulse time of 0.1 s and isolation power of 35%) and then subjected to IRMPD or sustained off-resonance irradiation (SORI)-CID. A gas pulse was programmed between the in-cell-isolation and IRMPD events; fragmentation of the radical species was then induced by IRMPD (50%, 0.5 s). For the SORI-CID experiments, the pulsed gas pressure was maintained at 8 mbar and the SORICID power was varied up to 5% with a pulse length of 0.2 s
Copyright © 2014 John Wiley & Sons, Ltd.
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RDD of peptides and proteins by IRMPD and SORI-CID with FTICR-MS and a frequency offset of –500 Hz. Up to 50 scans were averaged for each spectrum, and all spectra were calibrated internally with the precursor ion mass or externally with cesium iodide.
RESULTS AND DISCUSSION RDD of peptide RGYALG Figure 1 shows the 266 nm single laser pulse UVPD mass spectra obtained from both the FTICR and the LIT systems for the iodobenzoyl-modified peptide, RGYALG. The peak labeled as ’–Iodo’ represents a hydrogen-deficient radical [RGYALG]• generated by homolytic C–I bond cleavage. Side-chain losses (e.g., -43 L, -56 L, -106Y) and selective backbone fragmentation (i.e., a3) are secondary fragments that are spontaneously produced. Although the measured spectra from the two instruments show similar features, there are some differences in the fragmentation patterns that can be noted upon closer inspection. For example, the PD yield, which is quantified by the percentage of C–I bond cleavage given from formula (1), is greater in the LIT. The PD yield from the LIT (Fig. 1(a)) is 97%, which is close to the expected PD yield (~100%) for 4-iodobenzoyl chromophore under the optimal conditions. A marginally lower PD yield, 79%, is observed from the FTICRMS experiments (Fig. 1(b)). Identical lasers were used in the two experiments, but we were unable to obtain greater yields using FTICR-MS. There are several factors that may cause the decreased efficiency in the FTICR cell, including incomplete overlap of the ion cloud and laser beam, or reduced photon flux due to
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A second difference observed from Fig. 1 is the yield of secondary fragments (e.g., -43 L, -56 L, -106Y and a3) from RDD as defined by formula (2). The RDD yields from the LIT and the FTICR mass spectrometer are 22% and 35%, respectively. More secondary fragmentation is observed in the FTICR mass spectrometer, which is consistent with the expected trend given the pressure differences between the two instruments. More efficient collisional cooling of the ions is expected in the LIT, which leads to less spontaneous RDD than in FTICR-MS. Radical-directed dissociation yield ¼ X I all secondary fragments X 100% I all secondary fragments þ I radical peptide
(2) (2)
The fragmentation characteristics of the radical peptides were investigated further by utilizing additional excitation following re-isolation of the radical product ion (the m/z 739 ’-Iodo’ peak). Collisional activation in a LIT is shown in Fig. 2(a). Most of the observed product ions are the same as those secondary fragments that were measured in the PD experiment (cf. Fig. 1(a)), although some of the relative intensities differ. For example the loss of the tyrosine side chain (-106Y) is much more abundant relative to the leucine side-chain losses (-56L, -43L). A few minor secondary fragments are also observed in Fig. 2(a), which result from consecutive radical-initiated losses of small molecules (i.e., -43L-44CO2 and -56L-44CO2). For all consecutive losses, the radical remains on the peptide and even-electron fragments are ejected. In the FTICR cell, SORI-CID was used for additional collisional excitation. Significantly more fragments are observed from SORI-CID-FTICR MS (Fig. 2(b)) than from CID-LIT MS (Fig. 2(a)), and most of these fragments correspond to multiple consecutive losses of either side chains from tyrosine/leucine or CO2 from the C-terminus. The high mass accuracy of FTICRMS allows for confident assignment of these product ions (Table 1). It is clear from the results in Figs. 2(a) and 2(b) that SORI imparts significantly more energy into the molecule than collisional activation in the LIT. This is not surprising given that the pressure is much lower in the FTICR cell, necessitating that fewer collisions with higher energy occur to activate the ion. In addition, once the ions are activated, they are not collisionally cooled as occurs in the LIT, which will also favor the observation of sequential fragmentation product ions. IRMPD with FTICR-MS was also tested to provide supplemental activation to the radical peptide generated (Fig. 2(c)). The relative intensity of the precursor ion remained relatively high, yet many of the fragments have undergone multiple consecutive dissociation events. This suggests that
Copyright © 2014 John Wiley & Sons, Ltd.
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Figure 1. 266 nm UV photodissociation (UVPD) spectrum of peptide N-4-iodobenzoyl-RGYALG (m/z 739.4) in the 1+ charge state with (a) the linear ion trap (LIT) mass spectrometer and (b) the FTICR mass spectrometer. (The peak for the precursor ion is noted with a dashed arrow.)
beam divergence, as the laser pulse must travel much further to reach the ions. In terms of laser beam–ion cloud overlap, there are several potential causes. The ion cloud may be larger than the laser beam. Alternatively, the ion cloud may not sample the area covered by the laser pulse during the brief time that the laser is firing (~8 ns), i.e., it may be a temporal and spatial overlap problem. Further experiments will explore these possibilities. X I all fragments Photodissociation yield ¼ X 100% (1) I all peaks
X. Zhang et al. only part of the ion cloud is interacting significantly with the laser, enabling continuous activation of some ions while others experience little or no activation. Importantly, the intensity of the IR laser is significantly reduced in the twolaser arrangement relative to a dedicated IRMPD experiment because the silica window that transmits the UV light is not coated for optimal IR reflectivity. The IRMPD and SORI-CID spectra are fairly similar, suggesting that excited product ions were generated by similar pathways in both experiments. RDD of ubiquitin protein RDD of ubiquitin was examined on the two instruments and the results for the 9+ charge state are shown in Fig. 3. Collisional activation of the radical protein in the LIT yields very efficient fragmentation, leaving little relative intensity of the precursor ion (Fig. 3(a)). Although many fragments are easily generated, assigning them is difficult given the low resolving power (~1000) of the LIT. Analogous SORI-CID and
Figure 2. Tandem mass spectrum of radical peptide N-benzoyl-RGYALG in the 1+ charge state by (a) CID in the linear ion trap and (b) SORI and (c) IRMPD in the FTICR mass spectrometer. (The peak for the precursor ion is noted with a dashed arrow.)
Table 1. Mass deviations for fragments in Fig. 2(b)
Peaks
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-H2O -43L -44CO2 -56L -43L-44CO2 -56L-CO2 -106Y -106Y-43L -106Y-44CO2 -106Y-56L -106Y-43L-44CO2 -106Y-56L-44CO2
ppm relative Theoretical Experimental to theoretical m/z m/z m/z 721.3547 696.3105 695.3755 683.3027 652.3207 639.3129 633.3234 590.2687 589.3336 577.2608 546.2788 533.2710
721.3552 696.3113 695.3763 683.3037 652.3222 639.3146 633.3252 590.2710 589.3359 577.2634 546.2816 533.2739
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1 1 1 1 2 3 3 4 4 4 5 5
Figure 3. Tandem mass spectrum of radical ubiquitin in the 9+ charge state by (a) CID in linear ion trap and (b) SORI and (c) IRMPD in in the FTICR mass spectrometer. The average mass measurement accuracy for the FTICR-MS experiments was 1.5 ppm. (The peak for the precursor ion is noted with a dashed arrow.)
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RDD of peptides and proteins by IRMPD and SORI-CID with FTICR-MS IRMPD experiments in the FTICR mass spectrometer are shown in Figs. 3(b) and 3(c), respectively. In general, the fragmentation efficiency was lower in the FTICR mass spectrometer; however, the greatly increased resolution and mass accuracy allow for confident assignment of the product ions. One of the major reasons for the limited fragmentation efficiency for SORI-CID may be that the radical facilitates many low-energy dissociation pathways. Numerous attempts to improve the fragmentation efficiency by varying the SORICID power, pulsed gas length, or off-set frequency to increase activation led to unacceptable diminution of the overall ion count. The prevalence of products resulting from sequential losses is also minimal for this protein, suggesting that the larger product ions generated from a protein are not sufficiently activated to undergo additional fragmentation. Initial IRMPD experiments on radical ubiquitin yielded no product ions at all. Eventually, it was determined that in-cell isolation of the radical protein altered the ion cloud sufficiently that there was no overlap with the IR laser. To summarize, quadrupoleselected 9+ ubiquitin subjected to IRMPD immediately yielded abundant fragments, but the fragmentation efficiency decreased steeply if the same ions were additionally subjected to in-cell isolation. The degree of the loss of fragmentation efficiency correlated with the power of the in-cell isolation (Supplementary Fig. S1, Supporting Information). In order to ameliorate this issue and improve the overlap efficiency between the IR beam and the ion cloud, a short argon gas pulse (0.1–0.2 s) was applied after in-cell isolation to attempt to collisionally cool the ions back into the center of the cell. As can be seen in Fig. 3(c), a reasonable amount of product ions can be generated by IRMPD following the gas pulse. Another reason for the relatively low fragment intensity in Fig. 3 is that, in the RDD of proteins, side-chain fragmentations are very abundant (i.e., the big trailing peak after the radical protein precursor, as shown in Fig. 3) and thus may dilute other backbone fragmentation peaks. Most fragments from 9+ ubiquitin are multiply charged. Identification of these peaks is easily achievable for highresolution FTICR-MS but challenging in the low-resolution LIT. The charge state of individual peaks in Fig. 3(a) can be assigned using a slow scan mode for a narrow m/z region. However, even this approach is limited to abundant fragments due to constraints imposed by low signal-to-noise. In comparison, significantly more fragments are assigned in high-resolution FTICR-MS for both SORI-CID and IRMPD. All the radical-initiated backbone fragments in Figs. 3(a)–3(c) (e.g., z22, c54, d54, a59) are close in sequence to Y59, where the radical is initially created. This is consistent with limited radical migration along the protein. In addition, much less secondary fragmentation is observed in Figs. 3(b) and 3(c) than with the RDD of peptides in Figs. 2(b) and 2(c). Interestingly, there is no significant difference in fragmentation types among the three methods. As can be seen in Fig. 3, many of the major fragments (e.g., z22, c54, d54, a59) show up in all the three spectra, suggesting that both SORI-CID and IRMPD are appropriate methods for the RDD of proteins.
CONCLUSIONS
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Acknowledgements The authors gratefully acknowledge technical support from Jeremy Wolff (Bruker Daltonics) and funding from the National Institute of Health (R21GM103531 to RRJ and JAL, and R01GM103479 and S10RR028893 to JAL).
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The present results illustrate that a 266 nm UV laser can be successfully coupled to a 15-Tesla FTICR mass spectrometer for UVPD and RDD mass spectrometry experiments. The
PD yield from a single pulse in an FTICR mass spectrometer is influenced by the alignment efficiency between the UV photons and the ion cloud in the FTICR cell. The ultra-low-pressure environment in the FTICR cell makes collisional cooling less competitive, and thus more secondary fragments from RDD[29] are generated even in the absence of subsequent activation by SORI-CID or IRMPD. The major fragmentation pathways are the same among linear ion trap CID, SORI-CID and IRMPD. However, both peptide precursor ions and fragments are activated by SORI-CID and IRMPD. Therefore, consecutive dissociation is more prevalent in RDD spectra for FTICR-MS. Accurate identification of RDD fragments with FTICR-MS confirms previous experimental results obtained in a lowerresolution LIT. Compared to peptides, RDD of small proteins such as ubiquitin is difficult to perform by SORI-CID or IRMPD. For example, UVPD followed by IRMPD suffers from decreased alignment efficiency as a result of altered ion cloud shape, which is caused by in-cell isolation. Increasing the pressure by a gas pulse after in-cell isolation can effectively accelerate collisional cooling and improve IRMPD efficiency.
X. Zhang et al. [10] M. Lee, M. Kang, B. Moon, H. B. Oh. Gas-phase peptide sequencing by TEMPO-mediated radical generation. Analyst 2009, 134, 1706. [11] X. Zhang, R. R. Julian. Photoinitiated intramolecular diradical cross-linking of polyproline peptides in the gas phase. Phys. Chem. Chem. Phys. 2012, 14, 16243. [12] T. Ly, R. R. Julian. Elucidating the tertiary structure of protein ions in vacuo with site specific photoinitiated radical reactions. J. Am. Chem. Soc. 2010, 132, 8602. [13] J. K. Diedrich, R. R. Julian. Site-specific radical directed dissociation of peptides at phosphorylated residues. J. Am. Chem. Soc. 2008, 130, 12212. [14] S. Wee, R. A. J. O’Hair, W. D. McFadyen. Side-chain radical losses from radical cations allows distinction of leucine and isoleucine residues in the isomeric peptides Gly-XXX-Arg. Rapid Commun. Mass Spectrom. 2002, 16, 884. [15] M. C. Crowe, J. S. Brodbelt. Infrared multiphoton dissociation (IRMPD) and collisionally activated dissociation of peptides in a quadrupole ion trap with selective IRMPD of phosphopeptides. J. Am. Soc. Mass Spectrom. 2004, 15, 1581. [16] P. M. Remes, G. L. Glish. Mapping the distribution of ion positions as a function of quadrupole ion trap mass spectrometer operating parameters to optimize infrared multiphoton dissociation. J. Phys. Chem. A 2009, 113, 3447. [17] T. Y. Kim, M. S. Thompson, J. P. Reilly. Peptide photodissociation at 157 nm in a linear ion trap mass spectrometer. Rapid Commun. Mass Spectrom. 2005, 19, 1657. [18] J. A. Madsen, D. R. Boutz, J. S. Brodbelt. Ultrafast ultraviolet photodissociation at 193 nm and its applicability to proteomic workflows. J. Proteome Res. 2010, 9, 4205. [19] Sun, Q.Y., Julian, R.R.: Q. Y. Sun, R. R. Julian. Probing sites of histidine phosphorylation with iodination and tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2011, 25, 2240. [20] J. J. Wilson, J. S. Brodbelt. MS/MS simplification by 355 nm ultraviolet photodissociation of chromophorederivatized peptides in 4-3 quadrupole ion trap. Anal. Chem. 2007, 79, 7883.
[21] C. L. Kalcic, T. C. Gunaratne, A. D. Jonest, M. Dantus, G. E. Reid. Femtosecond laser-induced ionization/dissociation of protonated peptides. J. Am. Chem. Soc. 2009, 131, 940. [22] A. Racaud, R. Antoine, L. Joly, N. Mesplet, P. Dugourd, J. Lemoine. Wavelength-tunable ultraviolet photodissociation (UVPD) of heparin-derived disaccharides in a linear ion trap. J. Am. Soc. Mass Spectrom. 2009, 20, 1645. [23] D. P. Little, J. P. Speir, M. W. Senko, P. B. O’Connor, F. W. McLafferty. Infrared multiphoton dissociation of large multiply-charged ions for biomolecule sequencing. Anal. Chem. 1994, 66, 2809. [24] J. W. Flora, D. C. Muddiman. Determination of the relative energies of activation for the dissociation of aromatic versus aliphatic phosphopeptides by ESI-FTICR-MS and IRMPD. J. Am. Soc. Mass Spectrom. 2004, 15, 121. [25] H. J. Cooper, M. A. Case, G. L. McLendon, A. G. Marshall. Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of metal-ion selected dynamic protein libraries. J. Am. Chem. Soc. 2003, 125, 5331. [26] E. D. Dodds, J. B. German, C. B. Lebrilla. Enabling MALDIFTICR-MS/MS for high-performance proteomics through combination of infrared and collisional activation. Anal. Chem. 2007, 79, 9547. [27] Y. Hashimoto, H. Hasegawa, L. Waki. High sensitivity and broad dynamic range infrared multiphoton dissociation for a quadrupole ion trap. Rapid Commun. Mass Spectrom. 2004, 18, 2255. [28] B. E. Kemp, E. Benjamini, E. G. Krebs. Synthetic hexapeptide substrates and inhibitors of 3’-5’-cyclic amp-dependent protein-kinase. Proc. Natl. Acad. Sci. USA 1976, 73, 1038. [29] N. Leymarie, C. E. Costello, P. B. O’Connor. Electron capture dissociation initiates a free radical reaction cascade. J. Am. Chem. Soc. 2003, 125, 8949.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s website.
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 2729–2734
Revised: 1 October 2014
Accepted: 2 October 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 2729–2734 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7068
Radical-directed dissociation of peptides and proteins by infrared multiphoton dissociation and sustained off-resonance irradiation collision-induced dissociation with Fourier transform ion cyclotron resonance mass spectrometry Xing Zhang1, Huilin Li3, Benjamin Moore1, Piriya Wongkongkathep2, Rachel R. Ogorzalek Loo3,4, Joseph A. Loo2,3,4** and Ryan R. Julian1* 1
Department of Chemistry, University of California, Riverside, CA 92521, USA Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA 3 Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA 4 UCLA/DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095, USA 2
RATIONALE: Recent experiments utilizing photodissociation in linear ion traps have enabled significant development of Radical-Directed Dissociation (RDD) for the examination of peptides and proteins. The increased mass accuracy and resolution available in Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) should enable further progress in this area. Preliminary experiments with photoactivated radicals are reported herein. METHODS: A 266 nm Nd:YAG laser is coupled to a FTICR or linear ion trap mass spectrometer. Radical peptides and proteins are generated by ultraviolet photodissociation (PD) and further activated by collisions or infrared photons. RESULTS: A 266 nm UV laser and an IR laser can be simultaneously coupled to a 15 Tesla FTICR mass spectrometer. The ultra-low-pressure environment in FTICR-MS makes collisional cooling less competitive, and thus more secondary fragments are generated by UVPD than in linear ion traps. Activation by sustained off-resonance irradiation collisioninduced dissociation (SORI-CID) or infrared multiphoton dissociation (IRMPD) also yields additional secondary fragmentation relative to CID in an ion trap. Accurate identification of RDD fragments is possible in FTICR-MS. CONCLUSIONS: Relative to linear ion trap instruments, PD experiments in FTICR-MS are more difficult to execute due to poor ion cloud overlap and the low pressure environment. However, the results can be more easily interpreted due to the increased resolution and mass accuracy. Copyright © 2014 John Wiley & Sons, Ltd.
Bottom-up proteomics is widely used for protein identification and de novo protein sequencing.[1] Tandem mass spectrometry (MS/MS) can effectively verify the sequences of peptides with high confidence, speed and sensitivity, particularly with the aid of a high-performance mass spectrometer. Collisioninduced dissociation (CID) is the most widely used dissociation method for peptide/protein characterization due to its high efficiency and ease of implementation.[2] Electron-based dissociation methods, including electron-capture dissociation (ECD) and electron-transfer dissociation (ETD), have also proven to be effective in peptide sequencing and especially for the identification of post-translational modifications (PTMs).[3] The fragmentation types in these electron-based methods are complementary to the vibrational excitation dissociation pathways accessed by CID and thus can
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* Correspondence to: R. R. Julian, Department of Chemistry, University of California, Riverside, CA 92521, USA. E-mail: [email protected] ** Correspondence to: J. A. Loo, Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA. E-mail: [email protected]
potentially provide other useful structural information not available from CID. In contrast to even-electron dissociation pathways that dominate in CID, many of the fragments in ECD and ETD are generated by radical chemistries that initiate various homolytic bond cleavages. In addition to electron-based methods, radicals can also be generated on polypeptides in a more controlled manner through modification with radical precursors. Various radical precursors and methods have been developed to generate hydrogendeficient radical peptides in the gas phase, including oxidative dissociation of peptide-metal complexes,[4–6] and homolytic bond cleavage in modified groups such as nitrate ester,[7] peroxycarbamate,[8] nitroso,[9] and TEMPO.[10] These methods use CID to facilitate activation of radical precursors containing labile bonds that generate hydrogen-deficient radicals upon dissociation. In addition, photodissociation (PD) can be used for radical initiation, which offers the advantage of selective activation of specific chromophores.[11–13] Ultraviolet photodissociation (UVPD) at 266 nm has been used to selectively cleave carbon–iodine (C–I) or carbon–sulfur bonds that are present in chromophore-modified peptides and proteins or native sulfur–sulfur bonds present in disulfide bridges. After generation of radical peptides, further dissociation (either
X. Zhang et al. spontaneously or aided by collisional activation) leads to radicaldirected dissociation (RDD), including backbone fragmentation and side-chain loss. One typical feature in RDD is relatively abundant side-chain loss, which allows unambiguous identification of many specific amino acid residues and even distinction of isobaric residues such as leucine and isoleucine.[14] Both ion trap (linear and quadrupole) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers have the capability to trap ions for a flexible amount of time, which makes them ideal for coupling with lasers for photodissociation experiments. A variety of lasers with different wavelengths have been implemented on quadrupole ion trap[15–22] and FTICR mass spectrometers.[23–26] Although both instruments are capable of trapping ions, there are also important and obvious differences between them. For example, FTICR instruments are capable of achieving much higher resolution and mass accuracy than ion traps. There are also significant differences in pressure, with FTICR cells operating at 6–7 orders of magnitude lower pressure than ion traps. The lower pressure of the FTICR cell is advantageous for infrared activation of ions because there is minimal cooling.[27] On the other hand, FTICR cells are much larger than ion traps, require more time per scan, and they are located in the center of a large cryogenic magnet, which makes optimization of laser alignment including maximizing laser/ion cloud overlap significantly more challenging than interfacing lasers to linear ion traps. In the present work, 266 nm Nd:YAG lasers are coupled to both FTICR and linear ion trap (LIT) mass spectrometers. UVPD is performed on trapped ions in both instruments to cleave C–I bonds. The ions are then subjected to further activation by CID or infrared multiphoton dissociation (IRMPD) to investigate their subsequent radical chemistry. The differences in radical-directed dissociation between FTICR and LIT instruments are examined.
EXPERIMENTAL Materials A synthetic peptide with amino acid sequence RGYALG (residues 21-26 of chicken lysozyme and a synthetic peptide substrate for a cyclic-AMP dependent protein kinase[28]) was purchased from the American Peptide Company (Sunnyvale, CA, USA). Sodium iodide, chloramine-T, sodium metabisulfite and other organic solvents were purchased from Thermo Fisher Scientific (Waltham, MA USA). Bovine ubiquitin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Peptide derivatization Peptides were labeled at the N-terminus with N-hydroxysuccinimide (NHS)-activated iodobenzoyl ester (2× molar ratio) in 1:3 borate buffer (0.2 M, pH 8.5)/dioxane solution for 30 min at 40 °C. The side products at the arginine and tyrosine side chains were removed by incubation in 1 M hydroxylamine (adjust pH to 8–9 with NaOH) for 4 h. Protein iodination
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The tyrosine residue in ubiquitin (Y59) was iodinated with sodium iodide and chloramine-T at room temperature. After 10 min reaction time, excess sodium metabisulfite was added
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to quench the reaction. Stoichiometric quantities of reagents (1:2:1:2 molar ratio of peptide/sodium iodide/chloramine-T/ sodium metabisulfite) were used to limit the iodination extent and produce mainly mono-iodinated tyrosine. A reversed-phase cartridge (Michrom Bioresources, Inc., Auburn, CA, USA) was used for peptide and protein desalting and purification. The samples were washed with 2% acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA) and eluted with 80% ACN with 0.1% TFA. Photodissociation MS in linear ion trap instruments The polypeptides (10 μM) were dissolved in 50:50 H2O/CH3OH with 0.1% acetic acid and electrosprayed into an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). 266 nm photons were generated from the 4th harmonic of a Nd:YAG laser (Continuum, Santa Clara, CA, USA). The back plate of the instrument was modified with a quartz window to transmit UV photons into the linear ion trap. Laser pulses were synchronized to the isolation step via a digital delay generator. Photodissociation MS in FTICR-MS The experiments were performed using a 15-Tesla Bruker SolariX FTICR mass spectrometer with an infinity cell (Bruker Daltonics, Bremen, Germany). Data were acquired with an approximate resolving power of 400 000 at m/z 400. The electrospray ionization (ESI) capillary voltage was set to 4 kV. The temperature of dry nitrogen was set to 180 °C and the flow rate was kept at 2.5 L/min. The radiofrequency (RF) amplitude of the ion funnels was 200 Vpp, and the applied voltages were 100 V and 6 V for funnels 1 and 2, respectively. The voltage of skimmer 1 was 20 V and the skimmer 2 voltage was kept at 5 V. The RF frequencies used in all ion-transmission regions were multipole 1 (5 MHz), quadrupole (2 MHz), and transfer hexapole (2 MHz). Ions were accumulated for up to 20 s in the hexapole collision cell before being transmitted to the infinity ICR cell. The time-of-flight was set to 1.5 ms. The vacuum pressures for different regions were ~2 mbar for the source region, ~2 × 10–6 mbar for the quadrupole region, and ~2 × 10–9 mbar for the UHV-chamber pressure. IRMPD was performed with a Synrad 30 W CO2 laser (Mukilteo, WA, USA). Firing of the laser pulses (266 nm, Continuum Nd:YAG laser) was controlled by custom-programmed digital signals from the FTICR mass spectrometer. To align both UVPD and IRMPD lasers at the back of the infinity cell, a fused-silica window (Esco Optics, Oak Ridge, NJ, USA), positioned as a beam splitter, reflected the 10.6 μm beam 90° to the cell while transmitting the UV beam to the infinity cell. The experiment proceeded as follows: ions were filtered by m/z value in the quadrupole (Q1) and accumulated in the hexapole collision cell (h), then submitted to the infinity cell. One to ten UV pulses (10 Hz) homolytically cleaved carbon–iodine bonds. The radical species produced were isolated in the infinity cell (pulse time of 0.1 s and isolation power of 35%) and then subjected to IRMPD or sustained off-resonance irradiation (SORI)-CID. A gas pulse was programmed between the in-cell-isolation and IRMPD events; fragmentation of the radical species was then induced by IRMPD (50%, 0.5 s). For the SORI-CID experiments, the pulsed gas pressure was maintained at 8 mbar and the SORICID power was varied up to 5% with a pulse length of 0.2 s
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RDD of peptides and proteins by IRMPD and SORI-CID with FTICR-MS and a frequency offset of –500 Hz. Up to 50 scans were averaged for each spectrum, and all spectra were calibrated internally with the precursor ion mass or externally with cesium iodide.
RESULTS AND DISCUSSION RDD of peptide RGYALG Figure 1 shows the 266 nm single laser pulse UVPD mass spectra obtained from both the FTICR and the LIT systems for the iodobenzoyl-modified peptide, RGYALG. The peak labeled as ’–Iodo’ represents a hydrogen-deficient radical [RGYALG]• generated by homolytic C–I bond cleavage. Side-chain losses (e.g., -43 L, -56 L, -106Y) and selective backbone fragmentation (i.e., a3) are secondary fragments that are spontaneously produced. Although the measured spectra from the two instruments show similar features, there are some differences in the fragmentation patterns that can be noted upon closer inspection. For example, the PD yield, which is quantified by the percentage of C–I bond cleavage given from formula (1), is greater in the LIT. The PD yield from the LIT (Fig. 1(a)) is 97%, which is close to the expected PD yield (~100%) for 4-iodobenzoyl chromophore under the optimal conditions. A marginally lower PD yield, 79%, is observed from the FTICRMS experiments (Fig. 1(b)). Identical lasers were used in the two experiments, but we were unable to obtain greater yields using FTICR-MS. There are several factors that may cause the decreased efficiency in the FTICR cell, including incomplete overlap of the ion cloud and laser beam, or reduced photon flux due to
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A second difference observed from Fig. 1 is the yield of secondary fragments (e.g., -43 L, -56 L, -106Y and a3) from RDD as defined by formula (2). The RDD yields from the LIT and the FTICR mass spectrometer are 22% and 35%, respectively. More secondary fragmentation is observed in the FTICR mass spectrometer, which is consistent with the expected trend given the pressure differences between the two instruments. More efficient collisional cooling of the ions is expected in the LIT, which leads to less spontaneous RDD than in FTICR-MS. Radical-directed dissociation yield ¼ X I all secondary fragments X 100% I all secondary fragments þ I radical peptide
(2) (2)
The fragmentation characteristics of the radical peptides were investigated further by utilizing additional excitation following re-isolation of the radical product ion (the m/z 739 ’-Iodo’ peak). Collisional activation in a LIT is shown in Fig. 2(a). Most of the observed product ions are the same as those secondary fragments that were measured in the PD experiment (cf. Fig. 1(a)), although some of the relative intensities differ. For example the loss of the tyrosine side chain (-106Y) is much more abundant relative to the leucine side-chain losses (-56L, -43L). A few minor secondary fragments are also observed in Fig. 2(a), which result from consecutive radical-initiated losses of small molecules (i.e., -43L-44CO2 and -56L-44CO2). For all consecutive losses, the radical remains on the peptide and even-electron fragments are ejected. In the FTICR cell, SORI-CID was used for additional collisional excitation. Significantly more fragments are observed from SORI-CID-FTICR MS (Fig. 2(b)) than from CID-LIT MS (Fig. 2(a)), and most of these fragments correspond to multiple consecutive losses of either side chains from tyrosine/leucine or CO2 from the C-terminus. The high mass accuracy of FTICRMS allows for confident assignment of these product ions (Table 1). It is clear from the results in Figs. 2(a) and 2(b) that SORI imparts significantly more energy into the molecule than collisional activation in the LIT. This is not surprising given that the pressure is much lower in the FTICR cell, necessitating that fewer collisions with higher energy occur to activate the ion. In addition, once the ions are activated, they are not collisionally cooled as occurs in the LIT, which will also favor the observation of sequential fragmentation product ions. IRMPD with FTICR-MS was also tested to provide supplemental activation to the radical peptide generated (Fig. 2(c)). The relative intensity of the precursor ion remained relatively high, yet many of the fragments have undergone multiple consecutive dissociation events. This suggests that
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Figure 1. 266 nm UV photodissociation (UVPD) spectrum of peptide N-4-iodobenzoyl-RGYALG (m/z 739.4) in the 1+ charge state with (a) the linear ion trap (LIT) mass spectrometer and (b) the FTICR mass spectrometer. (The peak for the precursor ion is noted with a dashed arrow.)
beam divergence, as the laser pulse must travel much further to reach the ions. In terms of laser beam–ion cloud overlap, there are several potential causes. The ion cloud may be larger than the laser beam. Alternatively, the ion cloud may not sample the area covered by the laser pulse during the brief time that the laser is firing (~8 ns), i.e., it may be a temporal and spatial overlap problem. Further experiments will explore these possibilities. X I all fragments Photodissociation yield ¼ X 100% (1) I all peaks
X. Zhang et al. only part of the ion cloud is interacting significantly with the laser, enabling continuous activation of some ions while others experience little or no activation. Importantly, the intensity of the IR laser is significantly reduced in the twolaser arrangement relative to a dedicated IRMPD experiment because the silica window that transmits the UV light is not coated for optimal IR reflectivity. The IRMPD and SORI-CID spectra are fairly similar, suggesting that excited product ions were generated by similar pathways in both experiments. RDD of ubiquitin protein RDD of ubiquitin was examined on the two instruments and the results for the 9+ charge state are shown in Fig. 3. Collisional activation of the radical protein in the LIT yields very efficient fragmentation, leaving little relative intensity of the precursor ion (Fig. 3(a)). Although many fragments are easily generated, assigning them is difficult given the low resolving power (~1000) of the LIT. Analogous SORI-CID and
Figure 2. Tandem mass spectrum of radical peptide N-benzoyl-RGYALG in the 1+ charge state by (a) CID in the linear ion trap and (b) SORI and (c) IRMPD in the FTICR mass spectrometer. (The peak for the precursor ion is noted with a dashed arrow.)
Table 1. Mass deviations for fragments in Fig. 2(b)
Peaks
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-H2O -43L -44CO2 -56L -43L-44CO2 -56L-CO2 -106Y -106Y-43L -106Y-44CO2 -106Y-56L -106Y-43L-44CO2 -106Y-56L-44CO2
ppm relative Theoretical Experimental to theoretical m/z m/z m/z 721.3547 696.3105 695.3755 683.3027 652.3207 639.3129 633.3234 590.2687 589.3336 577.2608 546.2788 533.2710
721.3552 696.3113 695.3763 683.3037 652.3222 639.3146 633.3252 590.2710 589.3359 577.2634 546.2816 533.2739
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1 1 1 1 2 3 3 4 4 4 5 5
Figure 3. Tandem mass spectrum of radical ubiquitin in the 9+ charge state by (a) CID in linear ion trap and (b) SORI and (c) IRMPD in in the FTICR mass spectrometer. The average mass measurement accuracy for the FTICR-MS experiments was 1.5 ppm. (The peak for the precursor ion is noted with a dashed arrow.)
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RDD of peptides and proteins by IRMPD and SORI-CID with FTICR-MS IRMPD experiments in the FTICR mass spectrometer are shown in Figs. 3(b) and 3(c), respectively. In general, the fragmentation efficiency was lower in the FTICR mass spectrometer; however, the greatly increased resolution and mass accuracy allow for confident assignment of the product ions. One of the major reasons for the limited fragmentation efficiency for SORI-CID may be that the radical facilitates many low-energy dissociation pathways. Numerous attempts to improve the fragmentation efficiency by varying the SORICID power, pulsed gas length, or off-set frequency to increase activation led to unacceptable diminution of the overall ion count. The prevalence of products resulting from sequential losses is also minimal for this protein, suggesting that the larger product ions generated from a protein are not sufficiently activated to undergo additional fragmentation. Initial IRMPD experiments on radical ubiquitin yielded no product ions at all. Eventually, it was determined that in-cell isolation of the radical protein altered the ion cloud sufficiently that there was no overlap with the IR laser. To summarize, quadrupoleselected 9+ ubiquitin subjected to IRMPD immediately yielded abundant fragments, but the fragmentation efficiency decreased steeply if the same ions were additionally subjected to in-cell isolation. The degree of the loss of fragmentation efficiency correlated with the power of the in-cell isolation (Supplementary Fig. S1, Supporting Information). In order to ameliorate this issue and improve the overlap efficiency between the IR beam and the ion cloud, a short argon gas pulse (0.1–0.2 s) was applied after in-cell isolation to attempt to collisionally cool the ions back into the center of the cell. As can be seen in Fig. 3(c), a reasonable amount of product ions can be generated by IRMPD following the gas pulse. Another reason for the relatively low fragment intensity in Fig. 3 is that, in the RDD of proteins, side-chain fragmentations are very abundant (i.e., the big trailing peak after the radical protein precursor, as shown in Fig. 3) and thus may dilute other backbone fragmentation peaks. Most fragments from 9+ ubiquitin are multiply charged. Identification of these peaks is easily achievable for highresolution FTICR-MS but challenging in the low-resolution LIT. The charge state of individual peaks in Fig. 3(a) can be assigned using a slow scan mode for a narrow m/z region. However, even this approach is limited to abundant fragments due to constraints imposed by low signal-to-noise. In comparison, significantly more fragments are assigned in high-resolution FTICR-MS for both SORI-CID and IRMPD. All the radical-initiated backbone fragments in Figs. 3(a)–3(c) (e.g., z22, c54, d54, a59) are close in sequence to Y59, where the radical is initially created. This is consistent with limited radical migration along the protein. In addition, much less secondary fragmentation is observed in Figs. 3(b) and 3(c) than with the RDD of peptides in Figs. 2(b) and 2(c). Interestingly, there is no significant difference in fragmentation types among the three methods. As can be seen in Fig. 3, many of the major fragments (e.g., z22, c54, d54, a59) show up in all the three spectra, suggesting that both SORI-CID and IRMPD are appropriate methods for the RDD of proteins.
CONCLUSIONS
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Acknowledgements The authors gratefully acknowledge technical support from Jeremy Wolff (Bruker Daltonics) and funding from the National Institute of Health (R21GM103531 to RRJ and JAL, and R01GM103479 and S10RR028893 to JAL).
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The present results illustrate that a 266 nm UV laser can be successfully coupled to a 15-Tesla FTICR mass spectrometer for UVPD and RDD mass spectrometry experiments. The
PD yield from a single pulse in an FTICR mass spectrometer is influenced by the alignment efficiency between the UV photons and the ion cloud in the FTICR cell. The ultra-low-pressure environment in the FTICR cell makes collisional cooling less competitive, and thus more secondary fragments from RDD[29] are generated even in the absence of subsequent activation by SORI-CID or IRMPD. The major fragmentation pathways are the same among linear ion trap CID, SORI-CID and IRMPD. However, both peptide precursor ions and fragments are activated by SORI-CID and IRMPD. Therefore, consecutive dissociation is more prevalent in RDD spectra for FTICR-MS. Accurate identification of RDD fragments with FTICR-MS confirms previous experimental results obtained in a lowerresolution LIT. Compared to peptides, RDD of small proteins such as ubiquitin is difficult to perform by SORI-CID or IRMPD. For example, UVPD followed by IRMPD suffers from decreased alignment efficiency as a result of altered ion cloud shape, which is caused by in-cell isolation. Increasing the pressure by a gas pulse after in-cell isolation can effectively accelerate collisional cooling and improve IRMPD efficiency.
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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s website.
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